+

Nhx1

Aligning Orthologs and Identifying Alleles

Alexis Valauri-Orton and Puneet Lakhmani

+Why Nhx1? What does it do? Makes blueberries blue (Yamaguchi et al., 2001)

Controls vacuolar pH Anthocyanin expression is pH dependent

Pigmented anthocyanins cause berry color Elevated expression of Nhx1 causes bluing in flowers

Lack of Nhx1 prevents bluing

Vital to Salt Tolerance (Darley et al., 2000)

Key to Berry Ripening (Hanana et al., 2007) water uptake to the berry increases in berry size

+What can we learn from Nhx1 in blueberry? Determine how blueberries facilitate production of

healthful anthocyanins

Better understand ripening cycle A shift in the ripening cycle could make for blueberry wine

http://upload.wikimedia.org/wikipedia/commons/b/b9/Vaccinium_corymbosum_Beeren.jpg

+Nucleotide Errors Frameshifts

Latter half of hit inconsistent with other species in NCBI database

This hit picks up where the above hit “leaves off.”Aligns with other species in NCBI database

These hits are in two different reading frames+3+1

+

+1+3

N in sequence indicates potential error

Ignoring nucleotides N and A eliminates frameshift

We found the CaM binding domain within these two fragments If we had not corrected this frameshift error, we would not

have found this binding domain

Nucleotide Errors Frameshifts

+EST Data Unreliable

Hits discontinuous, fragmented

Aligned regions shift from frame to frame

+Identifying Families Initial Clustal W Hit:

Too many sequences Fragments align poorly with Grape and Arabidopsis, but alignment

patterns emerge

We identified 12 families of hits Hits within each family align closely

+All Families Displayed on Arabidopsis NHX1

+Family 1

4 alleles/paralogs/repeats

Equivalent of exon 11 in Arabidopsis

+Family 2

4 alleles/paralogs/repeatsEquivalent of exon 12

in Arabidopsis

+Family 3

3 alleles/paralogs/repeats

Equivalent of the latter half of exon 10 in Arabidopsis

+Family 4

5 alleles/paralogs/repeats Equivalent to the first half of exon 10

in Arabidopsis

+Family 5

4 alleles/paralogs/repeats

Equivalent of exon 8 in Arabidopsis

+Family 6

4 alleles/paralogs/repeats Equivalent of exon 9 in Arabidopsis

+Family 7

2 alleles/paralogs/repeats – imperfect alignment

Equivalent of end of exon 7,

beginning of exon 8 in Arabidopsis

+Family 8

4 alleles/paralogs/r

epeats

Equivalent of exon 6 in

Arabidopsis

+Family 9

5 alleles/paralogs/rep

eats

Equivalent of exon 1 in Arabidopsis

+Family 10

5 alleles/paralogs/r

epeats

Equivalent of latter half of

exon 2 in Arabidopsis

+Family 11

5 alleles/paralogs/repeats Equivalent of exon 3 in

Arabidopsis

+Family 12

5 alleles/paralogs/repeats – some poor alignment in

center

Equivalent of exon 13 in Arabidopsis

+Scaffolds Represented in Families

Scaffold Hits Across All Families

Nucleotides in Scaffold

01142 1 43,106

10492 5 9,573

03216 5 26,189

11152 5 8,889

01349 6 40,310

03649 6 24,321

01954 7 33,985

+All Families Displayed on Arabidopsis NHX1

+How many paralogs/alleles?

Difficult for us to determine now from our fragmented data

Allele numbers in other species give us some idea: Arabidopsis: only one Nhx1 listed in genome browser Grape: six putative Nhx1 genes listed in genome browser

We anticipate a number close to that of grapes Many fruits experience whole genome duplication Our data suggested variation/multiple copies of blueberry

Nhx1

+Further Steps

Wet lab work to extract entire gene PCR amplification using our data?

Find the promoter Myb binds to the Nhx1 promoter

Identify regulatory binding sites Properties of the blueberry may be explained by binding

sites on Nhx1 that are more or less sensitive than those in other species

Conduct more in-depth comparison of blueberry Nhx1 sequence and related orthologs

+Acknowledgements

Dr. Allan Brown and NC State University

The David H. Murdock Research Institute

Dr. Malcolm Campbell and Davidson College

+References

Darley, C.P., van Wuytswinkel, O.C., van der Woude, K., Mager, W.H., and de Boer, A.H. (2000). Arabidopsis thaliana and Sac- charomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na/H exchangers. Biochem. J. 351, 241–249.

Hanana M, Cagnac O, Yamaguchi T, Hamdi S, Ghorbel A, Blumwald E (2007) A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening. Plant Cell Physiol 48:804–811

Yamaguchi, T. e. a. (2001). Genes encoding the vacuolar Na+/H+ exchanger and flower coloration. Plant Cell Physiology, 42(5), 451--461.

Grape genome browser: http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/

Arabidopsis genome browser: http://atensembl.arabidopsis.info/index.html